Title

Human protein N-terminal acetyltransferase hNaa50p (hNAT5/hSAN) follows ordered sequential catalytic mechanism: combined kinetic and NMR study.

UMMS Affiliation

Department of Biochemistry and Molecular Pharmacology

Date

3-23-2012

Document Type

Article

Medical Subject Headings

Acetyl Coenzyme A; Acetyltransferases; Catalysis; Humans; Hydrophobic and Hydrophilic Interactions; Kinetics; Methionine; *Models, Chemical; Mutation; N-Terminal Acetyltransferase E; Nuclear Magnetic Resonance, Biomolecular; Peptides; Protein Conformation

Disciplines

Biochemistry | Enzymes and Coenzymes | Medicinal-Pharmaceutical Chemistry | Therapeutics

Abstract

N(alpha)-acetylation is a common protein modification catalyzed by different N-terminal acetyltransferases (NATs). Their essential role in the biogenesis and degradation of proteins is becoming increasingly evident. The NAT hNaa50p preferentially modifies peptides starting with methionine followed by a hydrophobic amino acid. hNaa50p also possesses N(epsilon)-autoacetylation activity. So far, no eukaryotic NAT has been mechanistically investigated. In this study, we used NMR spectroscopy, bisubstrate kinetic assays, and product inhibition experiments to demonstrate that hNaa50p utilizes an ordered Bi Bi reaction of the Theorell-Chance type. The NMR results, both the substrate binding study and the dynamic data, further indicate that the binding of acetyl-CoA induces a conformational change that is required for the peptide to bind to the active site. In support of an ordered Bi Bi reaction mechanism, addition of peptide in the absence of acetyl-CoA did not alter the structure of the protein. This model is further strengthened by the NMR results using a catalytically inactive hNaa50p mutant.

Rights and Permissions

Citation: J Biol Chem. 2012 Mar 23;287(13):10081-8. doi: 10.1074/jbc.M111.326587. Epub 2012 Feb 6. Link to article on publisher's site

Comments

At the time of publication, Paul Thompson was not yet affiliated with UMass Medical School.

Related Resources

Link to Article in PubMed